During Phase I, human Langerhans cell (LC) lines were obtained by transfecting or infecting LC progenitor cells derived from peripheral blood and bone marrow. The best cell lines were transformed with a well-known viral oncogene and could be expanded by 1000 fold in culture. A supplemental experiment showed that the cells could be cloned; in this non-optimized experiment, the most successful clone maintained on LC phenotype, exhibited log phase growth with a doubling time of 2.75 days, and had expanded 40,000 fold as of the date of submission of this proposal. Cryopreservation did not affect the ability of the cells to proliferate and the phenotype of the cells could be modulated with cytokines. The morphology of the cells remained dendritice and ultrastructural analysis confirmed that the cells contained Birbeck granules, the key ultrastructural feature of Langerhans cells (LC). Phenotypic characterization of cells showed that the cells could be matured into LC, as evidenced by high levels of CD1 a expression. In addition, in a functional assay, the cell lines retained their ability to stimulate T-cells in a mixed lymphocyte reaction (MLR). Phase II research will expand on the Phase I results. Initial studies will focus on the ability to further enhance the proliferate capacity and lifespan of the cells. The cells lines produced will be cloned and the phenotype, chemokine receptors, ultrastructure , gene expression and karotype of the resultant clones will be characterized. Chemotactic and phagocytic properties of the cells will be evaluated and the utility of the cell lines will be demonstrated in HIV infection and cancer immuno-therapy studies. Finally, the ability to incorporate the cells into a highly differentiated skin model and the capacity of such an immuno-competent model to predict allergic contact dermatitis will be studied.
The proposed research will produce human Langerhans cell lines useful in a broad spectrum of biomedical research. The LC lines will provide researchers the means to study the biology and function of LC in their native, unprimed state. Researchers will gain a valuable immunological tool to study and develop therapies against cancer and viral infections including HIV. Finally, a skin model containing immunologically competent cells would be extremely valuable to a large number of companies in evaluating whether their products are likely to cause allergic contact dermatitis.
